Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein...

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Bimolecular fluorescence complementation (BiFC) is a key technique for detecting protein-protein interactions in living cells.
• Existing BiFC systems have limitations in multicolor applications and signal characteristics.

Purpose of the Study:

• To develop and validate a novel red BiFC system based on mCherry for protein interaction studies.
• To demonstrate the utility of the red BiFC system for simultaneous detection of multiple protein interactions.

Main Methods:

• A red BiFC system was engineered by splitting the mCherry protein into two fragments.
• The system's functionality was verified using known interacting proteins: SV40 large T antigen (LTag) and human p53.
• The red BiFC system was combined with a Venus-based yellow BiFC system for simultaneous interaction detection.

Main Results:

• The new mCherry-based red BiFC system successfully detected the interaction between LTag and p53.
• Simultaneous detection of LTag-p53 and sp100-PML interactions was achieved in Vero cells using coupled red and yellow BiFC systems.
• The red BiFC system exhibits bright red fluorescence, rapid maturation, and favorable spectral properties.

Conclusions:

• The developed mCherry-based red BiFC system is a valuable tool for analyzing protein-protein interactions in living cells.
• This red BiFC system facilitates multicolor studies of protein interactions, enhancing the study of complex biological pathways.